16S RIBOSOMAL DNA AMPLIFICATION FOR PHYLOGENETIC STUDY
16S RIBOSOMAL DNA AMPLIFICATION FOR PHYLOGENETIC STUDY
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DOI：
10.1128/jb.173.2.697-703.1991
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发表时间：
1991-01-01
影响因子：
3.2
通讯作者：
LANE, DJ
中科院分区：
文献类型：
--
作者：
WEISBURG, WG;BARNS, SM;LANE, DJ
描述了一组能够在遗传学和分类学上广泛的细菌上引发酶扩增（聚合酶链反应）的寡核苷酸引物，沿着它们的使用方法和实例。 一对引物能够扩增来自许多细菌属的几乎全长16 S核糖体DNA（rDNA）;另外的引物可用于各种例外序列。 扩增材料的纯化方法，直接测序，克隆，测序和转录概述。 牛红细胞的专性细胞内寄生虫，边缘无浆体，被用作一个例子，其16 S rDNA扩增，克隆，测序，并进行基因定位。 无形体与立克次体属和埃立克体属有关。 此外，从美国典型培养物保藏中心的冻干安瓿中发现的材料中容易扩增来自几个物种的16 S rDNA。 利用该方法，可以在不需要培养的情况下进行极端苛养菌或高致病性菌的系统发育研究。 在理论上，任何可能设计聚合酶链反应引物的基因片段都可以从容易获得的冻干细菌培养物中获得。
A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.